Rotavirus (RV) is a leading global cause of childhood diarrhea. Current attenuated human RV (AttHRV) oral vaccines are effective in infants in developed countries, but for unexplained reasons, they lack efficacy in impoverished countries where diarrhea mortality is highest. Alternative affordable strategies are urgently needed to reduce diarrheal diseases and enhance oral vaccine efficacy. Malnutrition (kwashiorkor) and frequent antibiotic usage in infants affect the gut microbiome and barrier integrity, likely compromising gut immunity and predisposing infants to diarrheal illness, but their impact on HRV infection is uncharacterized. Studies of gnotobiotic (Gn) animals have revealed that probiotics or commensal microflora are crucial for gut immune maturation and homeostasis, but the mechanisms involved and their roles in modulating viral diarrheas or enhancing oral vaccine efficacy are unclear. Neonates undergo progressive changes in the gut microflora which we will model by colonizing Gn pigs with Lactobacillus spp (Gram, G+), E. coli (G- ), both, or a commensal cocktail from conventional piglets. Gn pigs resemble infants in size, anatomy, physiology, development of mucosal immunity and are the only animal model susceptible to HRV diarrhea. Our investigation of how the selected probiotics/commensals modulate gut homeostasis and immune responses and impact enteric diseases and vaccines will permit their rational use as biotherapeutic agents and/or adjuvants. We hypothesize that selected G+ and G- gut microflora will modulate different host cellular pathways leading to immunostimulatory, but balanced (Th1/Th2/Th17/Treg) responses that enhance efficacy of RV vaccines or immuno-regulatory (Treg) responses that moderate HRV diarrhea. Further, humanized, outbred Gn pigs are a unique model to study how microbiota and diet contribute to malnutrition and HRV disease severity under conditions that constrain confounding variables in ways not possible in infants. In Aim 1, we determine how selected G+ or G- probiotics, both, or the commensal cocktail modulates immune responses, gut homeostasis and HRV pathogenicity. Then we test the impact of antibiotics on the commensal microflora and on these same parameters. In Aim 2, we determine how defined probiotics or commensals modulate protective immunity to AttHRV oral vaccine. In Aim 3, in collaboration with Jeff Gordon, we develop a humanized Gn pig model to study the interaction of microbiota x diet (from African twins discordant for kwashiorkor) on severe malnutrition and HRV pathogenicity and identify microbial biomarkers to aid in vaccine design or therapeutic interventions. Effects of the probiotics/commensals/microbiota will be compared by intestinal transcriptome profiling (pig microarrays, 44K ESTs), metagenomics, metabolomics and metatranscriptomics, gut barrier integrity (sugar permeability, tight junction genes, serum LPS), and induction of innate and adaptive immune responses, to establish the immunoregulatory/immunostimulatory profiles. Our innovative studies will address gaps in knowledge of gut immune maturation and homeostasis and interactions between gut microflora and enteropathogenic viruses or oral vaccines. Our findings will contribute to alternative low cost probiotic treatments applicable to infants (or mothers) to moderate HRV disease, enhance oral vaccine efficacy, and reduce infant morbidity and mortality. PUBLIC HEALTH RELEVANCE: Human rotavirus (HRV) is a leading cause of childhood diarrhea worldwide. The high costs and unexplained low effectiveness of licensed oral HRV vaccines in infants in impoverished countries remain obstacles to their universal adoption. Alternative low cost treatments to moderate HRV disease and enhance effectiveness of RV and other oral vaccines are needed to reduce infant diarrheal diseases and deaths. A balanced, healthy gut microflora containing Gram positive (Lactobacillus sp) and Gram negative (E. coli) bacteria plays a crucial role in maturation of the neonatal intestinal and systemic immune systems. It is also critical for development of post-natal immune competence and resistance to infectious diseases or allergic and inflammatory bowel syndromes. Our goal is to use harmless probiotic or commensal microbes to confer beneficial effects to infant health via immune stimulation (pro-inflammatory strains to enhance vaccines) or immune regulation [anti-inflammatory strains to reduce inflammation (diarrhea, allergies, inflammatory bowel diseases)]. Our unique germfree (GF) pig model is derived free of confounding microbes and maternal antibodies. Neonatal GF pigs resemble infants in size, physiology, anatomy, susceptibility to HRV diarrhea and development of mucosal immunity. Results from our proposed studies will elucidate how a known Gram positive probiotic, a Gram negative commensal, or both, and a defined commensal mixture influence neonatal intestinal homeostasis and immune maturation, and consequently moderate HRV diarrhea or enhance effectiveness of attenuated HRV oral vaccines. Factors such as antibiotics and diet (malnutrition) can disrupt the gut microflora, influencing susceptibility to diarrheal diseases and the effectiveness of oral vaccines, but in undefined ways. Using the GF pig model, we will determine the impact of antibiotic treatment, commonly used in infants and children, on the gut microflora, neonatal immune responses and the severity of HRV diarrhea. With our collaborator, we will further develop a humanized GF pig model (transplanted microflora from African twins) to define the role of microflora and native diet on the development of severe forms of malnutrition such as kwashiorkor, seen in African children. We will then explore how severe malnutrition affects diarrhea and deaths associated with HRV, to discern the basis for the disproportionately high mortality rates in HRV-infected children in developing countries. Collectively these studies will identify potential microbial biomarkers that will aid in design of vaccines or therapeutic interventions, including dietary manipulations. Understanding how neonatal immune responses can be modulated in the context of probiotics and the gut microflora has important implications for public health. These include not only increasing neonatal immunity to viral vaccines and diarrheas, but also treatments for malnutrition and antibiotic-induced diarrheas, as well as for induction of tolerance to food antigens and our indigenous microflora, to control the increasing incidence of food allergies and inflammatory bowel syndromes, respectively.